Hepatitis C virus (HCV) is a major cause of liver disease, with about 130 million people infected worldwide. No vaccine has been developed so far, and the only treatment available today is a combination of pegylated interferon alpha and ribavirin. Progress in developing novel and specific treatments has been greatly hampered by the dearth of novel target proteins and suitable infectious HCV culture systems. We have developed high-throughput screening assays based on the inhibition of dimerization of HCV core, the viral capsid protein, which is essential for the assembly of the viral particle. These assays, now adopted by the pharmaceutical industry, have allowed us to identify novel inhibitors of HCV production. A validated hit of a screen based on our assays was used as an exquisite molecular probe to study the interaction of core with the NS3 helicase of HCV. It had however no effect on the interaction with NS5A, which we also characterized biochemically in our laboratory. We have therefore developed a new, bead-based transfer of energy assay to identify inhibitors of the core-NS5A interaction. The resulting compounds will constitute novel molecular probes for understanding how HCV non structural proteins participate in the assembly of the viral particle. These inhibitors may also serve as the basis of drugs to be combined with anti-viral treatments that should be more widely applicable than current single target or non-specific pharmacologic agents. PUBLIC HEALTH RELEVANCE: Hepatitis C affects 130 million individuals worldwide, 3 million of which live in the US, and the only treatment, a combination of interferon and ribavirin, is effective for less than half of the patients. We will develop a specific transfer-of energy-based assay screening assay for potent molecular probes of the virus, based on inhibition of interaction between core, the capsid protein essential for assembly of the viral particle, and NS5A, a HCV protein essential for viral replication. These compounds may lead to the development of orally available drugs for treatment of Hepatitis C.